Process for preparing processed sample liquid solution for electrophoresis

ABSTRACT

A process for preparing a processed sample liquid solution for gel electrophoresis, comprising (a) treating a sample comprising a cell suspension in a non-shearing manner to produce a processed sample liquid solution comprising a mixture of DNA fragments extracted from the cell suspension, wherein at least one of the DNA fragments is greater than 200 kilobase pairs and (b) transferring the processed sample liquid solution in a non-shearing manner directly to an electrophoresis medium for conducting electrophoresis.

FIELD OF THE INVENTION

This invention relates to the field of gel electrophoresis. Morespecifically, this invention relates to the preparation of a processedsample liquid solution, and to separation of DNA fragments of the sampleby electrophoresis, particularly pulsed-field gel electrophoresis.

BACKGROUND OF THE INVENTION

A major tool in molecular biology and related fields is electrophoreticseparation. In this process, mixtures of macromolecules, e.g., proteins,DNA, or RNA, are moved through a sieving medium, such as a gel, by anelectric field. Electrophoretic separation enables qualitative analysis,separation, recovery, and purification of macromolecules.

Nucleic acid molecules have one negative charge for each nucleotide. Asa result, the charge-to-mass ratio is constant for DNA of variouslengths and an electric field alone would not perform separation of DNA.In a porous medium, however, larger DNA molecules have greaterdifficulty traversing the enmeshing medium and move slower than smallerDNA molecules, thereby causing separation of DNA molecules of varyinglength. This separation effect is size limited, however, andconventional gel and capillary electrophoresis separation of DNAmolecules is generally limited to sizes smaller than 50 kilobase pairs(kbp).

U.S. Pat. No. 4,473,452 to Cantor teaches that electrophoresis of verylarge molecules can succeed by using alternating transverse electricfields. This method is known as pulsed-field gel electrophoresis (PFGE).It is generally believed that the chains of molecules presented to thePFGE gel are oriented and re-oriented by the fields, allowing the longerchains to traverse the gel medium without clogging the passages. Inaddition, the longer the chains are, the greater is the relaxation timeinvolved in the changes of orientation. As a result, the smaller chainsmove into and through the gel matrix much more readily than the largerchains. As described by E. Lai, et al. (“Pulsed-field GelElectrophoresis,” Bio Techniques, 7, pp. 34-42, 1989), the alternatingelectric fields can be applied in a number of orientations, giving riseto several variations of the PFGE method, e.g., field inversion gelelectrophoresis (FIGE), clamped homogeneous electric fields (CHEF)electrophoresis, and pulsed homogeneous orthogonal field gelelectrophoresis (PHOGE). The pulsed-field technique has also beenapplied to capillary electrophoresis for the separation of large DNAfragments as described by Kim and Morris (“Rapid Pulsed-field CapillaryElectrophoretic Separation of Megabase Nucleic Acids,” Anal. Chem. 67,pp. 784-786, 1995).

One of the primary applications of PFGE is the molecular typing ofbacteria. In this methodology, bacterial cells, imbedded in an agaroseplug, are treated to lyse the cells and remove or destroy cellularproteins. The released chromosomal DNA is then treated with arestriction endonuclease enzyme that cleaves infrequently to cut the DNAinto large fragments, typically between 50 and 800 kbp in size. Theselarge DNA fragments are separated using PFGE to yield a DNA fingerprintwhich can be used to identify the bacterium at the species andsub-species level and to differentiate among related bacteria.

While extremely effective for the molecular typing of bacteria and othermicrobes, PFGE has the major disadvantage of an extremely time-consumingsample treatment process, wherein three to four days is not uncommon.One reason the sample treatment process is time-consuming is the need toprotect the fragile DNA molecules from unintended mechanical breakage.Traditional methods for sample preparation in which the end product isDNA in solution are unsuitable for PFGE because large DNA molecules aresusceptible to shearing forces leading to mechanical breakage. JohnMaule, “Pulsed-Field Gel Electrophoresis”, Molecular Biotechnology,Volume 9, 1998, pp. 107-126.

The method of U.S. Pat. No. 4,473,452 to Cantor avoids mechanicalbreakage of the long DNA molecules by incorporating the bacterial cellsinto molded inserts (also known as “plugs”), typically made of anagarose gel, and performing the sample treatment on the entrapped cells.This sample treatment includes lysis of the bacterial cells, enzymaticdigestion of cellular proteins, and digestion of the DNA to producefragments of various sizes using an appropriate restriction endonucleaseenzyme. The treated plugs are then fitted into wells molded into theelectrophoresis gel and PFGE is performed, resulting in a fingerprintpattern for the bacterium. By using the plugs, the DNA molecules can beextracted from the cell and digested in a controlled manner, withoutunwanted mechanical breakage of the DNA.

Molecular typing of bacterial cells by PFGE has typically been achievedusing plugs. In this process, the cells or spheroplasts are suspended ingel (usually agarose) and then poured into molds to form the plugs. Thesample treatment steps of lysis, deproteinization, and digestion areperformed on the cells, embedded in the agarose plugs, as follows.First, the sample plugs are placed in a solution containing a lysingagent, e.g., lysozyme, and incubated at the appropriate temperatureovernight. The lysing solution is then removed and the plugs are washedwith buffer. A protease-containing solution, e.g., Proteinase K, is nextadded to digest proteins and the plugs are incubated overnight at theappropriate temperature. The next day, the protease solution is removed,and the plugs are washed several times with wash buffer. The plugs arewashed with diluted wash buffer and then with the restriction enzymebuffer. A suitable restriction endonuclease-containing solution is thenadded to the plugs and the plugs are incubated overnight at theappropriate temperature. The next day, the restriction endonucleasesolution is removed, and the plugs are washed with the wash buffer. Afinal wash is performed with the electrophoresis buffer, e.g., 0.5×tris-borate-EDTA (TBE) buffer. The plugs are then inserted into matchingwells formed in a gel slab by a suitable comb and the pulsed-fieldelectrophoretic separation is carried out. Care must be taken at everystep to ensure that the plugs are not damaged in the process.

This sample plug treatment protocol avoids mechanical breakage inhandling long and fragile DNA molecules. However, it makes sampletreatment very tedious and time consuming, and the results are operatordependent. Sufficient time is required for diffusion of reagents intothe agarose plugs. Moreover, the use of plugs makes automation of thesample preparation process very difficult, if not impossible. It isbelieved that there are no known reports of an automated sampletreatment process for bacterial typing using PFGE.

Some practitioners have evolved one day protocols for PFGE employingplugs. But these protocols are often labor intensive and require highlyskilled personnel. For example, Turabelidze, et al. (“ImprovedPulsed-Field Gel Electrophoresis for Typing Vancomycin-ResistantEnterococci,” J. Clinical Microbiology November 2000, p. 4242-4245)describes a rapid protocol for sub-typing vancomycin-resistantenterococci in approximately one day. Gautom (“Rapid Pulsed-Field GelElectrophoresis Protocol for Typing Of Escherichia coli 0157:H7 andOther Gram-Negative Organisms in 1 Day,” J. Clin. Microbiol. 35,November 1997, pp. 2977-2980) teaches a standardized protocol that isdone in one day using bacterial cells directly from the culture plates,shortening cell lysis and deproteinization, using preheated buffer, andshorter restriction digestion times. In these more rapid methods,nevertheless, plugs are employed.

It has been reported in the CHEF-DR® II Pulsed-field ElectrophoresisSystems Instruction Manual and Applications Guide from Bio-RadLaboratories that liquid samples can be transferred and separated usingPFGE when working with DNA in the size ranging from 50 kbp up to 200 kbpby taking special precautions not to mechanically break the DNAmolecules. Specifically, the use of a pipet tip with a large opening isrecommended. There is believed to be no known report of a non-shearingtreatment and transfer of a liquid sample having DNA lengths greaterthan 200 kbp. For bacterial typing, the DNA fragments of interest oftenare greater than 200 kbp and typically range from 50 kbp to 1000 kbp,and are even greater than 1000 kbp in some instances. Therefore, forbacterial typing, sample plugs have been required to prevent mechanicalbreakage of the DNA molecules during sample treatment and gel loading.

There remains a need to reduce the sample treatment time required in themolecular typing of bacteria using PFGE. In addition, there is a need toeliminate the use of sample plugs in PFGE without causing unwantedmechanical breakage of the DNA molecules. There also is a need to treatand transfer bacterial samples for PFGE with a minimum degree ofoperator dependence by automating the sample treatment process.

SUMMARY OF THE INVENTION

The present invention provides a process for preparing a processedsample liquid solution for electrophoresis that eliminates the use ofgel plugs. The processed sample liquid solution comprises a mixture ofDNA fragments wherein at least one of the DNA fragments is greater than200 kilobase pairs. The process comprises the steps of:

-   -   (a) treating a sample comprising a cell suspension in a        non-shearing manner to produce a processed sample liquid        solution comprising a mixture of DNA fragments extracted from        the cell suspension, wherein at least one of the DNA fragments        is greater than 200 kilobase pairs; and    -   (b) transferring the processed sample liquid solution in a        non-shearing manner directly to an electrophoresis medium for        conducting electrophoresis.

The invention also provides a method for separating a mixture of DNAfragments from a sample. This method comprises the steps of:

-   -   (a) treating a sample comprising a cell suspension in a        non-shearing manner to produce a processed sample liquid        solution comprising a mixture of DNA fragments extracted from        the cell suspension, wherein at least one of the DNA fragments        is greater than 200 kilobase pairs;    -   (b) transferring the processed sample liquid solution in a        non-shearing manner directly to an electrophoresis medium for        conducting electrophoresis; and    -   (c) separating the mixture of DNA fragments by conducting        electrophoresis.

In one embodiment of the invention, the DNA fragments are 50 kbp to 1000kbp. In another embodiment, at least one of the DNA fragments is greaterthan 225 kilobase pairs, is greater than 250 kilobase pairs, or isgreater than 300 kilobase pairs.

The cell suspension can comprise one or more cells that are suspended ina lysis buffer. In one embodiment, the cell suspension is a bacterialcell suspension.

The step of treating the cell suspension can comprise the steps ofsubjecting the cell suspension to lysis, deproteinization, anddigestion. The treatment step can be automated or manual. In theautomated treatment step, the treatment of the cell suspension,including reagent additions and incubations, is achieved using anautomated apparatus. The step of transferring the processed sampleliquid solution to the well of the electrophoresis gel can be automatedor manual.

The electrophoresis medium can be an electrophoresis gel, including theelectrophoresis gels used for pulsed-field gel electrophoresis. Theelectrophoresis medium can also be a viscous sieving solution other thana gel, including the viscous solutions used for pulsed-field capillaryelectrophoresis. In a preferred embodiment, the processed samplesolution is transferred to a well of the pulsed-field electrophoresismedium.

BRIEF DESCRIPTION OF THE DRAWING

The invention is better understood from the following detaileddescription of preferred embodiments of the invention when read inconnection with the accompanying drawing. It is emphasized that,according to common practice, the various features of the drawing arenot to scale, unless otherwise indicated herein. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawing are the following figures:

FIG. 1 is an elevational drawing of a pipet that can be used in theprocess of the invention.

FIG. 2 is a cross sectional drawing of a sample carrier suitable for theinvention.

FIG. 3A to FIG. 3D are sequential, perspective views of an apparatusassembled to carryout the process of the invention.

FIG. 4 shows the image of an agarose gel with the electrophoreticpattern of bacterial DNA from Example 1 obtained using optimizedpipetting parameters.

FIG. 5 shows the image of an agarose gel with the electrophoreticpattern of bacterial DNA from Example 2 comparing conventional sampleplugs with processed liquid samples.

FIG. 6 shows the image of an agarose gel with the electrophoreticpattern of bacterial DNA from Example 3 obtained using optimizedpipetting parameters.

FIG. 7 shows the image of an agarose gel with the electrophoreticpattern of bacterial DNA from Example 4 obtained while determiningoperating conditions to minimize mechanical breakage of the DNA.

FIG. 8 shows the image of an agarose gel with the electrophoreticpattern of bacterial DNA from Example 5 obtained using the defaultRiboPrinter® System pipetting parameters.

FIG. 9 shows the image of an agarose gel with the electrophoreticpattern of bacterial DNA from Example 6 obtained using low force manualpipetting.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Described herein is a process for treatment of a sample comprising acell suspension, such as a bacterial cell suspension, and delivering theresulting processed sample liquid solution to an electrophoresis medium.This methodology is particularly useful for the automation of sampletreatment for molecular typing of bacteria using pulsed-field gelelectrophoresis (PFGE).

Contrary to standard operating procedures for sample treatment ofbacterial cells for molecular typing using PFGE as hitherto performed,it has been discovered that the prepared molecular chains of DNA (“DNAmolecules”) can be handled in a liquid medium, unsupported by a gelmatrix such as gel plugs. In accordance with the invention, the cellsuspension is treated to produce a processed sample liquid solutioncomprising a mixture of DNA fragments wherein at least one of thefragments is greater than 200 kbp. Both the treatment of the cellsuspension, which includes lysis, deproteinization, and digestion, andthe transfer of the resulting processed sample liquid solution to theelectrophoresis medium are undertaken in a non-shearing manner, since itis critical that the DNA molecules do not become fragmented due tounintended mechanical breakage.

By a “non-shearing manner” is meant that the forces acting on the DNAmolecules during both treatment of the cell suspension and transfer ofthe processed sample liquid solution to the electrophoresis medium areminimized to prevent mechanical breakage of the DNA molecules.Mechanical breakage means that the DNA molecules are randomly fragmentedor sheared by physical forces exerted upon the molecules. These forcesinclude shear, tensile, and torsional forces. This mechanical breakageis distinctly different than the very specific DNA cleavage that iscatalyzed by restriction endonuclease enzymes, which results in thedesired DNA fragment pattern. If mechanical breakage of the DNA doesoccur, such breakage can be detected on the gel image following theseparation of the restriction fragments by electrophoresis, such gelimage having a loss of DNA bands and the appearance of a smear in thesample lanes.

The forces causing mechanical breakage of DNA molecules and the degreeof sensitivity of the DNA molecules to such forces are readily apparentto those skilled in the art. Typically, larger DNA molecules are morefragile or delicate and tend to mechanically break more readily thansmaller fragments. In the examples provided herein, the DNA size rangeutilized was between 50 kbp and 700 kbp since this size range is typicalof that encountered in the molecular typing of bacteria.

One factor that particularly influences DNA breakage is the viscosity ofthe solutions delivered to treat the sample, and the viscosity of theprocessed sample liquid solution itself. Higher viscosity results in ahigher force during reagent delivery and mixing and, therefore,increases DNA mechanical breakage. The viscosity of the reagentsolutions used in the examples was between 1.17 and 3.44 cP(centipoise), measured at 25° C.

Another factor influencing mechanical breakage of the DNA is thegeometry of the system, which includes the inner diameter and geometryof the pipet tip that delivers the reagents to the sample and theprocessed sample liquid solution to the electrophoresis gel, the sizeand shape of the sample well used to contain the sample during theaddition of the lysis, deproteinization, and digestion reagents, and theposition of the pipet tip in the sample well during reagent delivery.The geometry of the system can be configured, in conjunction with otherfactors that potentially cause mechanical breakage, to prevent DNAbreakage. For example, a pipet tip with a larger inner diameter willtypically result in less DNA breakage than a pipet tip with a smallinner diameter.

A schematic representation of a pipet tip 2 that can be used in thepresent invention is shown in FIG. 1. In one embodiment of theinvention, the tip has a length of 141.0 mm +/−0.25 mm, a tip innerdiameter (ID) of 0.50 mm +/−0.10 mm below the tapered region 4, and anID of 1.60 mm +/−0.10 mm above the tapered region. The geometry of asuitable sample well 6 is depicted in FIG. 2. In the examples detailedbelow, the pipet tip was positioned beneath the surface of the liquid inthe center of the sample well, approximately 3-4 mm from the bottom ofthe well, for all of the reagent deliveries.

Yet another factor influencing mechanical breakage of DNA molecules isthe velocity and acceleration of reagent delivery and sample mixing. Thevelocity and acceleration must be controlled to prevent DNA breakage.

The invention includes the step of (a) treating a sample comprising acell suspension in a non-shearing manner to produce a processed sampleliquid solution comprising a mixture of DNA fragments extracted from thecell suspension, wherein at least one of the DNA fragments is greaterthan 200 kilobase pairs. This treating step can be conducted followingmethods of extracting DNA in situ taught in U.S. Pat. No. 5,595,876 toRakestraw, hereby incorporated by reference in its entirety.

These methods include the steps of:

-   -   (i) thermally treating the cells of the microorganism to        deactivate the endogenous nuclease activity associated with the        cells;    -   (ii) lysing the microorganism cells to release the component DNA        and incubating the DNA to render the DNA amenable to subsequent        enzymatic action; and    -   (iii) thermally treating the DNA to denature remaining nucleases        and proteases thereby deactivating any residual nuclease and        protease activity.

This treating, as taught in U.S. Pat. No. 5,595,876 to Rakestraw, canoptionally further include the step of (iv) further enzymaticmodification of the DNA in the same reaction vessel. Followingextraction, the DNA is then enzymatically digested in situ. One exampleis the digestion of the lysate with the restriction endonuclease EcoRIto generate a spectrum of DNA fragments.

This method, which includes (A) lysis to break open the cells, (B)deproteinization to destroy cellular proteins, and (C) digestion (with arestriction enzyme) to cut the DNA into fragments, is referred to hereinas “LDD.”

In the automated pipetting examples below (Examples 1-4), the reagentdelivery parameters were optimized using the lysis, deproteinization,and digestion (LDD) module of the RiboPrinter® MicrobialCharacterization System available from Qualicon, Inc. All mixing stepswere eliminated and the velocity and acceleration parameters for reagentdelivery were reduced from the default parameters of the RiboPrinter®System. Example 5 is a comparative example that demonstrates mechanicalbreakage of the DNA when the default RiboPrinter® System pipettingparameters were used.

Reagent mixing in the process of the invention occurs via the agitationresulting from reagent delivery and by diffusion. Although the sampleliquid could be mixed gently by aspiration, this increases the chancesof mechanical breakage of the DNA and it has been discovered that suchmixing was unnecessary. The optimum parameters for reagent delivery weredetermined empirically by varying the reagent delivery velocity andacceleration rates and observing the mechanical breakage that resultedon the gel image following electrophoresis, as described in Example 4.In Example 4, it was discovered that a reagent delivery velocity of 38μL/sec and acceleration of 200 μL/sec² gave consistently low DNAbreakage. Higher velocities, up to 581 μL/sec with an acceleration of12800 μL/sec², could be used, but the results were not consistent, i.e.,mechanical breakage was observed on some runs, but not on others. Thetransfer of the processed sample liquid solution to the electrophoresismedium is a critical step in the process because it is the only timethat the DNA is aspirated into a pipet tip. The parameters used in thetransfer of the sample liquid solution to the electrophoresis medium canbe determined by routine experimentation.

The term “electrophoresis medium” includes those media used forelectrophoretic separation. Electrophoresis media, as used herein,includes electrophoresis gels and non-gel viscous solutions that providethe sieving properties necessary to conduct electrophoretic separation.

Manual Process Treatment of Sample

As demonstrated below in Example 6, it was also discovered thatsubstantially non-shearing conditions can be achieved by manualpipetting using a very slow delivery speed. In this case, the reagentdeliveries were made to the sample well with the pipet tip placedagainst the side of the well above the sample to level. Although goodresults can be obtained with the manual technique, it is onerous andrequires a patient operator, so it is recognized that the process isbest performed by automated or mechanical means to insurereproducibility, ease of operation, and mechanical integrity of the DNAfragments.

The manual process can be performed as follows. First, bacterial cellsare grown on an appropriate agar medium such as brain heart infusion(BHI) agar. Next, cells that have formed a solid lawn in the incubatedagar medium are picked using an autoclaved colony pick and aretransferred into a microcentrifuge tube held in a sample rack. The cellsare then mixed with sample buffer (e.g., a hypotonic solution having anEDTA concentration between about 5 mM and 30 mM) with a vortexer.

The number of picks needed to give the optimum cell concentration can bedetermined empirically or by measuring the cell concentration by methodswell known in the art, e.g., an optical density measurement or bycounting cells using a hemocytometer. 30 μL of the sample liquid is thenadded to each well of the RiboPrinter® System sample carrier. Next, thesample carrier is placed in the heat treatment station of theRiboPrinter® System, which functions as a heating block to heat thesamples to a temperature of about 80±2° C. for about 10 min todeactivate endogenous nuclease activity and to kill the cells.

After the samples cool, the sample carrier is loaded onto theRiboPrinter® System, which serves as a computer-controlled heating blockfor the manual process. Then, the lysing agent achromopeptidase (ACP) isadded to each sample by very slowly pipetting the solution down the sideof the sample wells. In this way, the sample is not agitated, and nomechanical breakage of the DNA results. It is possible to mix thesamples by very slowly aspirating the sample liquid into the pipet tipand then returning the sample liquid to the sample well by pipettingdown the wall using a pipet tip that has been cut-off to yield a largeropening, although such mixing is not required. ACP, in addition toserving as the lysing agent, also serves as the deproteinization reagentbecause it possesses proteolytic activity in addition to its lyticactivity. The samples are then incubated at a suitable temperature,generally 37° C., for a time sufficient to lyse the cells and digestcellular proteins, typically 20 min to one hour. After this incubation,the samples are heated 70° C. to 75° C. for 15 min to 30 min toinactivate the ACP. Then, the appropriate restriction endonucleaseenzyme is added to the samples in the manner described above. Thesamples are incubated at a suitable temperature for the restrictionenzyme used. The incubation time depends on the restriction enzymeactivity and the type of sample. Typically, the incubation time rangesfrom 1 hour to 4 hours, although longer times can be used, if necessary,to obtain complete digestion of the DNA.

Loading buffer is then manually pipetted slowly down the side of thesample wells to densify the samples for gel loading. The samples aremixed gently as described above. The samples are then added to the wellsof the electrophoresis gel using the gentle pipetting method describedabove. The PFGE can then be run according to standard procedure.

Automated Process Treatment of Sample

Although treatment of the sample can be done manually, it is preferableto automate treatment steps to obtain the most reproducible results.FIG. 3A to FIG. 3D consecutively show a method in accordance with U.S.Pat. No. 5,595,876 to Rakestraw as applied to the instant invention,using an apparatus adapted from that of the aforementioned RiboPrinter®System. What cannot be seen, of course, is the restructured softwarethat ensures minimal mechanical breakage of the DNA molecules. Althoughthe process using the RiboPrinter® System is described for purposes ofillustration, one skilled in the art will recognize that anycomputer-controlled solution delivery apparatus with temperature controlcould be used to carry out the automated process of the instantinvention.

In FIG. 3A, a sample 10 is picked from petri dish 12 using pick 14 andtransferred to a tube 16, containing a hypotonic, EDTA-containing buffersolution, held in rack 18 shown in FIG. 3B. The sample liquid is mixedusing a vortexer 20 such as the Biovortexer obtainable from BiospecProducts of Bartlesville, Okla. Referring next to FIG. 3C, the sampleliquid is transferred using pipet 22 to one of the wells 24 in samplecarrier 26 held in heat treatment station 28. A suitable heat treatmentstation may be obtained from Qualicon, Inc. The wells 24 are capped toprevent evaporation. The heat treatment station 28 is activated andperforms the time and temperature regimen, described above, todeactivate nucleases and inactivate the cells.

The sample carrier 26 is then placed in a cavity 27 in sample prep unit30 as shown in FIG. 3D. Cavity 31, which is in line with cavity 27 andseparated by wash unit 32, includes a DNA prep pack 34. The DNA preppack contains all the reagents required for the lysis, deproteinization,and digestion treatment, including an ACP tablet, buffer for therestriction enzyme, and loading buffer. All these reagents are containedin wells sealed by a cover 38 which is made from a material that isreadily pierced, such as foil. The appropriate restriction enzyme iscontained in vial 36, which is inserted into an opening in the DNA preppack. Also in line is traveler 40 which is controllably traversed alonga line in accordance with arrows X-X by means not shown along a controlarm 42 between the sample prep unit 30 and the gel box 48 containing agel 44 up to a gel line 46. The gel box 48 and sample prep unit 30 canbe fixed together by an arm 50. Within traveler 40 is a pipet, also notshown, which moves controllably in the direction indicated by the arrowsY-Y orthogonal to X-X. These motions are computer directed. The pipet isin fluid communication with sources of wash fluid and pumping means toaspirate and eject regulated small amounts of fluid.

In use, after the carrier 26 is loaded into cavity 27, the systemcontrolling traveler 40 is actuated. The pipet is moved over the well inthe prep pack that contains the ACP and a volume of water is added tore-hydrate the reagent. The solution is mixed by aspiration. Then, aselected aliquot of the ACP solution is aspirated into the pipet.Traveler 40 then moves over a selected well 24 in carrier 26, moves downto pierce the cap, and injects the aliquot into the well 24. The rate ofinjection is selected to minimize the force on the fluid handled toprevent mechanical breakage of the DNA molecules. Traveler 40 is thenmoved over wash station 32, the pipet is lowered and the inside andoutside of the pipet tip is cleansed. This process is repeated for eachsample. The samples are incubated in the presence of the ACP solutionfor a time sufficient to lyse the cells and inactivate internal cellulardegradative factors, typically 20 min to 60 min. The samples are thenheated to a temperature between 70° C. and 75° C. for a time sufficientto inactivate the remaining protease and nuclease activity,approximately 30 min. The samples are then cooled to the appropriatetemperature for the restriction enzyme digestion.

The traveler is then moved over the appropriate well in prep pack 34 anddownwardly through the cover 38 and into the selected buffer (at a 10×concentration) for the restriction enzyme. The buffer is then added tothe vial containing the restriction enzyme and the solutions are mixedby aspiration. Typical restriction enzymes used for the molecular typingof bacteria using pulsed-field gel electrophoresis include, for example,Xbal, Sfil, Smal, Notl, Apal, and Ascl. The enzyme selected controlsdigestion to produce DNA fragments having a length from about 50 kbp toabout 800 kbp.

The restriction enzyme is then carried back to the selected well 24 andinjected at a rate controlled to limit mechanical breakage of the DNAmolecules. The samples are not mixed after the addition of the reagents.The wash and aspirate routine is repeated as needed until allingredients required are added to the wells 24. In one embodiment, eightsamples may be worked on in order. The wells 24 are then subjected to acontrolled time at a selected temperature by means not shown. Finally,the loading buffer solution is added to each sample, as described abovefor the preceding reagents, to densify the samples to enable loadinginto the submerged gel. Then traveler 40 is actuated to remove a portionof the processed sample liquid solution from the wells 24 and inject, ina non-shearing manner, the contents into selected molded-in wells in theelectrophoresis gel which are located in the line of traversal oftraveler 40. Alternatively, this sample transfer to the electrophoresisgel can be done by gentle, manual pipetting. The electrophoresis gel issubmerged in an electrophoresis buffer, e.g., 0.5× tris-borate-EDTA(TBE) buffer, during loading of the samples. Suitable pipet washingbetween well attendance is carried out to prevent cross-contamination ofthe samples.

Thus, the step of treating the sample can be performed in a singlereaction vessel in an apparatus that is part of the RiboPrinter®Microbial Characterization System available from Qualicon, Inc.,Wilmington, Del. The apparatus for automated sample treatment forpulsed-field gel electrophoresis of the invention can comprise acomputer-directed automated apparatus for performing, in a substantiallynon-shearing manner, lysis, deproteinization and digestion, inaccordance with processes of Rakestraw in U.S. Pat. No. 5,595,876, on asample liquid comprising a bacterial cell suspension of the bacterialtarget in a lysis buffer to produce a processed sample liquid solution,containing fragments of the DNA of said target; said preparativeapparatus optionally operatively associated with computer-directed,automated, liquid-transfer apparatus for transferring the said processedsample liquid solution in a substantially non-shearing manner to atleast one well prepared in a gel slab in an associated, pulsed-field gelelectrophoresis apparatus.

In one embodiment, the heat treatment station is separated from the mainapparatus of the RiboPrinter® System. Separating the heat treatmentstation from the main apparatus of the RiboPrinter® System permitsconfining the handling of viable pathogens to a biological safety level2 (BSL2) laboratory (“BioSafety in Microbiological and BiomedicalLaboratories,” U.S. Department of Health and Human Services, CDC/HIN,U.S. Government Printing Office; 2^(nd) ed., May, 1988 HHS PublicationNo. (NIH) 88-8395) and permits conveniently locating the majorprocessing steps in general laboratory space.

EXAMPLES

In the examples, the reagent delivery and sample transfer parameterswere optimized based on the specified conditions. For other DNA fragmentsizes, reagent viscosities, and system geometries, these reagentdelivery and sample transfer parameters can be determined by routineexperimentation, as described above.

Example 1

Automated Treatment of Bacterial Cells Using Optimized PipettingParameters

In this example, all the pipetting, with the exception of the initialpreparation of the cell suspension and the loading of the processedsample liquid solutions into the electrophoresis gel, was done in anautomated manner using a separate lysis, deproteinization, and digestion(LDD) module from the RiboPrinter® Microbial Characterization System. Anew LDD protocol was written specifically for the PFGE sample treatment,wherein the software was modified to reduce the speed at which thereagents were delivered to the samples to minimize forces that causemechanical breakage of DNA molecules. Specifically, the reagent deliveryvelocity was decreased from 375 μL/s to 38 μL/s, the acceleration wasdecreased from 1250 μL/s² to 200 μL/s², and all sample mixes wereeliminated. Furthermore, the initial incubation at 45° C. in thestandard RiboPrinter® System protocol was eliminated and the ACPincubation step was increased from 25 min to 45 min at 37° C.

E. coli, ATCC strain 51739, was cultured on brain heart infusion (BHI)agar overnight in an incubator at 37° C. The next day, colonies werepicked using a RiboPrinter® System colony pick, available from Qualicon,Inc. Five picks were suspended in 300 μL of RiboPrinter® System samplebuffer and the suspension was mixed using a vortexer. Thirty microlitersof the cell suspension was added manually to each of the eight wells ofthe RiboPrinter® System sample carrier. The sample carrier was placed inthe heat treatment station, supplied with the RiboPrinter® System, andheated at 80° C. for 10 min to inactivate the cells and to deactivateendogenous nuclease activity. When the sample carrier cooled after theheat treatment station treatment, it was loaded into the LDD Module. TheDNA prep pack was modified by removing the digestion tablet andreplacing it with 294 μL of 10× NEB Buffer 2 containing 10× BSA, bothobtained from New England BioLabs, Inc. Fifty microliters of Xbal (20Units/μL), purchased from New England BioLabs, Inc., was added to anempty RiboPrinter® System restriction enzyme vial, which was insertedinto the DNA prep pack. The prep pack was loaded into the LDD module toinitiate the LDD process.

The LDD module re-hydrated the ACP tablet and delivered 70 μL of theresulting solution to each sample. The samples were incubated at 37° C.for 45 min. After this time, the samples were heated to 70° C. for 25min. Next, the LDD pipet removed 243 μL of the 10× restriction enzymebuffer (Buffer 2 plus BSA) and added it to the restriction enzyme vialcontaining the Xbal and the solution was mixed by aspiration. Then, theLDD pipet delivered 23 μL of the resulting Xbal solution to each sample,and the samples were incubated at 37° C. for three hours. After thistime, 10 μL of the RiboPrinter® System loading buffer (low saltformulation) was added to each sample well by the LDD pipet and thesample carrier was removed from the module.

The 1% agarose gel for the pulsed-field electrophoresis run was preparedin 0.5× tris-borate-EDTA (TBE) buffer according to the manufacturer'sdirections using pulsed-field grade agarose (purchased from Bio-RadLaboratories). A 10 well comb, 14 cm wide and 0.75 mm thick, was used toform the wells in the gel. After the gel was prepared, sample lanes 1and 10 were filled with plugs containing the lambda ladder DNA sizemarker, obtained from Bio-Rad Laboratories. This DNA size markerconsists of concatemers of lambda DNA so that the fragments increase insize in approximately 48 kbp increments. The gel was then placed in thegel box of the Bio-Rad CHEF-DRII pulsed-field electrophoresis system,which was filled with 0.5× tris-borate-EDTA (TBE) buffer.

The processed liquid sample solutions were loaded into theelectrophoresis gel using manual pipetting as follows. The samplesolutions were mixed once very slowly by aspiration using a 200 μLGilson Pipetman® to disperse the loading buffer and then added veryslowly to wells 2 through 9 using the same Gilson Pipetman®. Therecirculating pump of the PFGE apparatus was not turned on untilapproximately 30 minutes after the electrophoresis was started to allowthe DNA molecules to enter the gel. This was done so the liquid sampleswould not be washed out of the wells.

The gel was run for 21 hours on the Bio-Rad CHEF-DRII pulsed-fieldelectrophoresis system using a field strength of 6 V/cm, with initialand final switch times of 1 sec and 35 sec, respectively. After the run,the gel was removed from the gel box and placed in a solution containingethidium bromide (0.5 μg/mL in water) for approximately one hour. Thegel was then incubated in a tray containing Milli-Q water forapproximately two hours to de-stain the gel. The gel was placed into theBio-Rad Gel-Doc 1000 system to obtain an image of the gel, which isshown in FIG. 4.

As can be seen from FIG. 4, the reproducibility of the DNA patterns isexcellent and no evidence of mechanical breakage is observed, asevidenced by the sharp bands and the absence of any in-lane smear.Mechanical breakage did not occur because of the gentle pipettingtechniques that were used to minimize the forces acting on the DNAmolecules.

Example 2

Processed Sample Liquid Solution of the Invention Compared withConventional Plug

In this example, conventional sample plugs were compared with theprocessed liquid sample solution of the invention using several strainsof E. coli. The strains used were E. coli ATCC No. 51739, E. coli0157:H7 strain ATCC No. 43889, and E. coli 0157:H7 strain 43894. Thesestrains were cultured on BHI agar overnight at 37° C.

The processed sample liquid solution of the invention was prepared andtransferred to an electrophoresis gel as described in Example 1. Theconventional plugs were prepared according to published protocol usingthe CHEF bacterial genomic DNA plug kit obtained from Bio-RadLaboratories. The cultures were sampled by taking 4 colony picks off theagar plates and placing the picks in 500 μL cell suspension buffer fromthe Bio-Rad kit. These cell suspensions and a 2% agarose solution wereplaced in a water bath thermostated at 50° C. The sample plugs wereprepared by adding 500 μL of the agarose solution to 500 μL of each ofthe cell suspensions. The samples were mixed by aspiration using a pipetand were placed back into the water bath. Then, one plug mold (tenplugs) was prepared for each sample by adding 100 μL of the appropriatesample mixture to each well of one plug mold and the mold was placed inthe refrigerator at 4° C. for 15 min. In this way, ten plugs were madefor each strain being tested. The 10 sample plugs for each strain wereremoved from the molds and placed into 50 mL centrifuge tubes containing5.0 mL of lysozyme solution (0.2 mL of Lysozyme, 2.5 mg/mL, in 5.0 mL ofLysozyme buffer). A screened cap was placed on each tube to facilitatesolution removal. The plugs were incubated in the lysozyme solutionovernight at 37° C.

The next day, the tubes containing the plugs were placed in an ice bathfor 15-20 min to harden the plugs. Then, the lysozyme solution wasdrained from the tubes through the screened caps and 25 mL of 1× washbuffer was added. The tubes were rocked gently on a rocker platform for10-15 min. The wash solution was drained and 2.5 mL of Proteinase Kreaction buffer was added to each tube. One hundred microliters ofProteinase K was then added to each tube and the tubes were incubatedovernight at 50° C.

The tubes were placed in an ice bath for 15-20 min. Next, the ProteinaseK solution was drained from the tubes through the screened caps and 25mL of 1× wash buffer was added to each tube. The tubes were then placedon a rocker platform and were rocked gently for one hour. After thistime, the wash solution was removed and this wash process was repeatedthree more times to give a total of four washes. Then, 10 mL of 1× washbuffer was added to each tube. One plug was removed from each tube andthe plugs were placed in separate 50 mL centrifuge tubes containing 25mL of 0.1× wash buffer. The tubes were placed on a rocker platform androcked gently for 30 min. The plugs were then transferred to separate1.5 mL centrifuge tubes and 900 μL of 1× restriction enzyme buffer (NEBBuffer 2 containing 1× BSA) was added to each tube. The tubes wereplaced on the rocker platform for one hour. The buffer was removed fromeach tube using a pipet and 300 μL of fresh 1× restriction enzyme bufferwas added to each tube. Then, 10 μL of Xbal restriction enzyme was addedto each tube and the tubes were incubated at 37° C. overnight.

The next day, the tubes were placed in an ice bath for 15-20 min. Therestriction enzyme solution was removed from each tube using a pipet andthe plugs were washed with 1.0 mL of 1× wash buffer for 30 min on therocker platform. This wash solution was removed from the tubes using apipet and replaced with 1.0 mL of 0.5× TBE buffer and the tubes wererocked for 30 min.

The agarose gel was prepared as described in Example 1 and sample lane 1was filled with a plug containing the lambda ladder DNA size marker.Then, the sample plugs were loaded into the gel as follows: ATCC StrainNo. 51739 in lane 2; ATCC Strain No. 43889 in lane 5; and ATCC StrainNo. 43894 in lane 8. The gel was placed into the gel box of the Bio-RadCHEF-DRII pulsed-field electrophoresis system, which was filled with0.5× tris-borate-EDTA (TBE) buffer. The processed sample liquidsolutions were loaded manually, as described in Example 1, in the twowells adjacent to the plug samples, i.e., ATCC Strain No. 51739 in lanes3 and 4, ATCC Strain No. 43889 in lanes 6 and 7, and ATCC Strain No.43894 in lanes 9 and 10. The PFGE conditions were the same as given inExample 1. The results of this experiment are shown in FIG. 5. As can beseen, the patterns obtained for the liquid samples are identical tothose obtained with the conventional sample plugs.

Example 3

Automated Treatment of Several E. coli 0157:H7 Strains Using OptimizedPipetting Parameters

Eight strains of E. coli 0157:H7, isolated from several outbreaks, werecultured, treated, and transferred to an electrophoresis gel asdescribed in Example 1. The results are shown in FIG. 6. As can be seen,eight distinct patterns were obtained and no mechanical breakage of theDNA was observed, as evidenced by the sharp bands and the absence of anyin-lane smear.

Example 4

Determination of Operating Conditions to Minimize DNA MechanicalBreakage

Our original concept was that the long, processing-time deficiency ofconventional methods could be overcome by working in a liquid mediumentirely and that the problems of the past in breaking long DNAmolecules in liquid handling could be avoided in the proper low forceregime. That this concept was sound is shown by the previous examples.In this example, optimum reagent delivery parameters were experimentallydetermined using the RiboPrinter® System LDD module.

The process used with bacterial samples for automated pulsed-field gelelectrophoresis analysis is given below. A diagram showing thetransition region of the pipet tip is provided in FIG. 1, and thedimensions of the pipet tip used to deliver the reagents are as follows:Length 141.0 +/− 0.25 mm (5.551 +/− 0.010 in) Tip ID  0.50 +/− 0.10 mm(0.020 +/− 0.004 in) ID after transition  1.60 +/− 0.10 mm (0.062 +/−0.004 in)

The geometry of a suitable sample well 6 is depicted in FIG. 2.

The wells of the sample carrier were initially filled with 30 μL of asuspension of bacterial cells in buffer, as described in Example 1. Thisstep was done using manual pipetting. The sample carrier was then heatedon the heat treatment station at 80° C. for 10 min. Some evaporationoccurred during this step, reducing the sample volume to approximately22 μL.

The sample carrier was then placed into the LDD Module and the processbegun. The instrument was programmed to add 70 μL of an enzyme solutionin buffer to each well. There was an incubation period, followed by aheat step at 70° C. for 25 min. This step was followed by a cool downperiod to return the temperature to 37° C. The second reagent additionwas then made, consisting of an addition of 23 μL of an enzyme solution.This enzyme solution contained 8.5% glycerol, thus having a higherviscosity than an aqueous buffer, i.e., 1.64 cP at 25° C.

Next, another incubation was conducted, followed by addition of 10 μL ofloading buffer, which is a fairly viscous solution. The actual viscosityof the loading buffer was 3.44 cP at 25° C.

For each reagent addition, the pipet tip was positioned between 3 mm and4 mm from the bottom of the well.

After the last reagent addition, the samples were removed manually usingvery gentle pipetting and were added to the wells of the gel, asdescribed in Example 1. The addition parameters that were tested for allthree reagents are given below in Table I. TABLE I Reagent AdditionReagent Addition Sample Well Velocity (μL/s) Acceleration (μL/s²) 1 38200 2 116 2000 3 193 3800 4 271 5600 5 348 7400 6 426 9200 7 503 11000 8581 12800The velocity-time profile in our system is trapezoidal. The velocitiesgiven above are the maximum velocities.

The results from this experiment are shown in FIG. 7. As can be seen inFIG. 7, no significant mechanical breakage of the DNA was evident insamples 1-6. The parameters for sample 7 gave significant mechanicalbreakage of the DNA fragments, as evidenced by the loss of bands and thein-lane smear. The sample 8 parameters gave almost total degradation ofthe DNA in the size range of interest (50 to 70 kbp) due to mechanicalbreakage. Therefore, controlling the reagent addition parameters to beless than those used in Sample 6 above will minimize the forces thatcause mechanical breakage of the DNA molecules during sample treatment.The results of this experiment were not consistent. On some runs nomechanical breakage of DNA was observed at the highest reagent additionvelocities and accelerations used, i.e., samples 7 and 8. Theseinconsistent results probably resulted from the normal variability ofthe automated process. For example, small changes in the position of thesample carrier, relative to its placement in the LDD module, result invariability of the pipet position relative to the sample wells duringreagent delivery. This variability affects the flow patterns in thewells and therefore, the forces acting on the DNA molecules. However,the low reagent delivery and acceleration parameters used for sample 1,which are preferred, always gave good results, i.e., no significantmechanical breakage of the DNA molecules.

Example 5

Automated Treatment of Bacterial Cells Using Default RiboPrinter® SystemPipetting Parameters

This Example was done as described in Example 1, except that the defaultRiboPrinter® System pipet delivery velocity and acceleration were used,i.e., reagent delivery velocity of 375 μL/s with an acceleration of 1250μL/s². The samples were also mixed several times by aspiration afterreagent delivery. The results are shown in FIG. 8. As can be seen, therewas significant degradation of the DNA caused by mechanical breakage, asevidenced by the loss of all bands above about 150 kbp and the in-lanesmear below 150 kbp. This example demonstrates the need to carefullycontrol the forces induced by reagent delivery and mixing.

Example 6

Manual Treatment of Bacterial Cells Using Low Force Pipetting

In the following example, all the pipetting was done manually. TheRiboPrinter® System lysis deproteinization digestion (LDD) module wasused only as a computer-controlled heating block. The pipet on theinstrument was inactivated.

E. coli, ATCC strain 51739, was cultured on brain heart infusion (BHI)agar overnight in an incubator at 37° C. The next day, colonies werepicked using a RiboPrinter® System colony pick. Five picks weresuspended in 300 μL of RiboPrinter® System sample buffer and thesuspension was mixed using a vortexer. Thirty microliters of the cellsuspension was added to each of the eight wells of the RiboPrinter®System sample carrier. The sample carrier was placed in the heattreatment station, supplied with the RiboPrinter® System, and heated to80° C. for 10 min to inactivate the cells and to deactivate endogenousnuclease activity.

The RiboPrinter® System was started by selecting a modified substituteenzyme batch (SEC batch), which had an extended digestion time, i.e.,37° C. for 180 min, from the operations pull-down menu and the batch wassubmitted to the instrument. The achromopeptidase (ACP) tablet in theRiboPrinter® System DNA prep pack was rehydrated with 990 μL of Milli-Qwater and the solution was mixed by vortexing. After the sample carrierreturned to room temperature after heat treatment station treatment, 70μL of the ACP solution was added to each sample well using a 100 μLGilson Pipetman®. The solution was added very slowly down the side ofthe sample wells to minimize the forces on the DNA molecules. The samplesolutions were not mixed. The sample carrier was placed into the LDDmodule of the RiboPrinter® System and the other consumables were loaded,according to the manufacturer's directions, to begin the LDD process.

At the start of the LDD process, the samples were incubated at 42° C.for 15 min and then, at 37° C. for an additional 25 min to lyse thecells and digest cellular proteins. The samples were then heated to 70°C. for 25 min to deactivate the ACP enzyme. The restriction enzymesolution was prepared as follows. Fifty microliters of Xbal (20Units/μL), purchased from New England BioLabs, Inc., was added to anempty RiboPrinter® System restriction enzyme vial. Then, 243 μL of 10×NEB Buffer 2 containing 10× BSA (both obtained from New England BioLabs,Inc.) was added to the vial containing the Xbal and the solution wasmixed by repeated aspiration. After the samples cooled down followingprotease deactivation, twenty-three microliters of the Xbal solution wasadded to each sample using a 100 μL Gilson Pipetman®. The solution wasadded very gently down the side of the well and the samples were notmixed to minimize the forces on the DNA molecules. The samples wereincubated at 37° C. for three hours. After this time, 10 μL of theRiboPrinter® System loading buffer (low salt formulation) was added toeach sample well by slowly pipetting the solution down the side of thewell to minimize the forces on the DNA molecules. The samples were notmixed. The sample carrier was then removed from the RiboPrinter® Systeminstrument and the batch was terminated manually.

The samples were loaded manually into the gel and the electrophoreticseparation was run as described in Example 1. The resulting gel image isshown in FIG. 9. As can be seen in the figure, sharp bands with littleevidence of mechanical breakage of the DNA were obtained.

Although illustrated and described above with reference to specificembodiments, the present invention is nevertheless not intended to belimited to the details shown. Rather, various modifications may be madein the details within the scope and range of equivalents of the claimsand without departing from the spirit of the invention.

1. A process for preparing a processed sample liquid solution for electrophoresis, comprising the steps of: (a) treating a sample comprising a cell suspension in a non-shearing manner to produce a processed sample liquid solution comprising a mixture of DNA fragments extracted from said cell suspension, wherein at least one of said DNA fragments is greater than 200 kilobase pairs; and (b) transferring said processed sample liquid solution in a non-shearing manner directly to an electrophoresis medium for conducting electrophoresis.
 2. The process of claim 1 wherein said cell suspension comprises one or more cells suspended in a lysis buffer.
 3. The process of claim 1 wherein said cell suspension is a bacterial cell suspension.
 4. The process of claim 1 wherein said treating comprises subjecting said cell suspension to lysis, deproteinization, and digestion.
 5. The process of claim 4 wherein said lysis and deproteinization comprises treatment with the enzyme achromopeptidase.
 6. The process of claim 4 wherein said digestion comprises treatment with a restriction enzyme.
 7. The process of claim 6 wherein said restriction enzyme is selected from the group consisting of Xbal, Sfil, Smal, Notl, Apal, and Ascl.
 8. The process of claim 1 wherein said DNA fragments are 50 kilobase pairs to 1000 kilobase pairs.
 9. The process of claim 1 wherein said step (a) is automated.
 10. The process of claim 1 wherein said step (b) is automated.
 11. The process of claim 1 wherein said steps (a) and (b) are automated.
 12. The process of claim 1 wherein said electrophoresis medium is an electrophoresis gel.
 13. The process of claim 1 wherein said electrophoresis medium is a well of an electrophoresis gel.
 14. The process of claim 1 wherein said electrophoresis medium is a viscous sieving solution.
 15. A process for separating a mixture of DNA fragments extracted from a cell suspension, comprising the steps of: (a) treating a sample comprising a cell suspension in a non-shearing manner to produce a processed sample liquid solution comprising a mixture of DNA fragments extracted from said cell suspension, wherein at least one of said DNA fragments is greater than 200 kilobase pairs; (b) transferring said processed sample liquid solution in a non-shearing manner directly to an electrophoresis medium; and (c) separating said mixture of DNA fragments by conducting electrophoresis.
 16. The process of claim 15 wherein said cell suspension comprises one or more cells suspended in a lysis buffer.
 17. The process of claim 15 wherein said cell suspension is a bacterial cell suspension.
 18. The process of claim 15 wherein said DNA fragments are 50 kilobase pairs to 1000 kilobase pairs.
 19. The process of claim 15 wherein said step (a) is automated.
 20. The process of claim 15 wherein said step (b) is automated.
 21. The process of claim 15 wherein said steps (a) and (b) are automated.
 22. The process of claim 15 wherein said electrophoresis medium is an electrophoresis gel and said electrophoresis is pulsed-field gel electrophoresis.
 23. The process of claim 15 wherein said processed sample liquid solution is transferred to a well of said electrophoresis medium.
 24. The process of claim 15 wherein said electrophoresis is pulsed-field capillary electrophoresis.
 25. The process of claim 15 wherein said treating comprises subjecting said cell suspension to lysis, deproteinization, and digestion.
 26. The process of claim 25 wherein said lysis and deproteinization comprises treatment with the enzyme achromopeptidase.
 27. The process of claim 25 wherein said digestion comprises treatment with a restriction enzyme.
 28. The process of claim 27 wherein said restriction enzyme is selected from the group consisting of Xbal, Sfil, Smal, Notl, Apal, and Ascl. 